CN107475401A - The method and primer of food-borne bacillus cereus are detected using loop-mediated isothermal amplification technique - Google Patents
The method and primer of food-borne bacillus cereus are detected using loop-mediated isothermal amplification technique Download PDFInfo
- Publication number
- CN107475401A CN107475401A CN201710804909.6A CN201710804909A CN107475401A CN 107475401 A CN107475401 A CN 107475401A CN 201710804909 A CN201710804909 A CN 201710804909A CN 107475401 A CN107475401 A CN 107475401A
- Authority
- CN
- China
- Prior art keywords
- bacillus cereus
- primer
- detected
- food
- loop
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method and primer that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique.Methods described includes：Extract the DNA of detected sample；Using described DNA as template, LAMP reactions are carried out using described primer；Reaction product is identified judge whether contain bacillus cereus in detected sample.For the conserved sequence design primer of bacillus cereus virulence gene, a pair of LAMP reaction systems of going forward side by side optimize for invention, can stablize, high specific, highly sensitive detection bacillus cereus.
Description
Technical field
The present invention relates to the detection of food-borne pathogenic microorganism, more particularly to one kind to be examined using loop-mediated isothermal amplification technique
The method for surveying food-borne bacillus cereus.
Background technology
The detection method for bacterium mainly has bacteria distribution culture identification method, immunological detection method, pathology at present
Tissue section method and molecular biology method.
Bacteria distribution culture identification application fluid nutrient medium, semisolid culturemedium or solid medium will be isolated
Doubtful bacterium is cultivated, and application hemolytic experiment, motility are tested, produce the biochemical tests such as sour aerogenesis and dyeing microscopic examination to micro- life
Thing is diagnosed.At present, China it is domestic most common and it is most widely used be National Standard Method (GB4789.30-94), also have it
His isolation and identification method such as FDA (food and medicine Surveillance Authority of the U.S.) method, improvement FDA methods, rower method (SN) and GNFIS methods
Deng.Accuracy of this kind of method in current all types of detection methods is highest, but either which kind of is separately cultured
Technology, they have one it is common the shortcomings that, exactly from sample collect pathogenic microorganism separation qualification result, it is necessary to
Test period all can be very long, and the culture of general microorganism needs 2 days or so, and the required time of identification and result are in a star
Or so phase, some microorganisms are separately cultured needs more than ten days, and what is just obtained goes out result.Therefore, such method can not adapt to mesh
Preceding fast and accurately diagnostic requirements market.Simultaneously because during operation, the bacterium of live body is contacted, operating personnel are formed
Certain health threat.
The principle of immunological detection method is that the compound of precipitation is combined to form according to Ag-Ab, common method bag
Include agglutinating reaction method, precipitation reaction process, immunofluorescence technique, radioimmunoassay method, EUSA, benefit
Body binding tests etc..Immunological detection method is a kind of serum that the principle combined based on antigen with antibody specificity is detected
Method.The shortcomings that this diagnostic method be in detection process, it is necessary to reagent it is very expensive, while the method is in detection process
In, according to different detection objective microbes, its specificity is also not quite similar, false positive results easily occurs, cause to judge by accident.Together
Influence of the Shi Renwei operation for experimental result is also what is had, so needing the laboratory profession with certain quality and ability
Personnel.
Histopathologic slide's method is to take a certain size pathological tissues, and pathological section is made with histopathological methods
(generally pathological tissues are embedded in paraffin mass, thinly sliced with slicer, then are dyed with hematoxylin-eosin (H-E)), uses
Microscope further checks the lesion of tissue.According to the occurrence and development process of lesion, pathological diagnosis is finally made.This detection side
Although method accuracy is very high, it is maximum the problem of be the professional requirement meeting of the personnel to being engaged in Tissue pathological diagnosis
It is very high, it is necessary to professional Histopathology teacher or be engaged in the postgraduate of Histopathology research, be possible to carry out accurate
True diagnosis, it may also be said to which this kind of diagnostic method needs the experiment people on microscopic pathology is gained knowledge with abundant experience and experience
Member.
Molecular biology is the subject for studying biological phenomena material base on a molecular scale.Common is micro- applied to detecting
Biological method have the gel electrophoresis experiment of nucleic acid (DNA), the molecule hybrid experiment of nucleic acid, nucleic acid amplification in vitro experiment etc. side
Method, it is the specific ideal of detection method the advantages of these methods, detection time can most be completed in 6 hours soon.This
The shortcomings that method, is alternating temperature consersion unit and detecting instrument, it is necessary to expensive.Molecular biological testing is in experimentation
Middle to need complicated variable temperature unit, the price of this instrument is very high, and the dispensing for basic unit is using being difficult to accomplish all standing.
Bacillus cereus food poisoning has obvious seasonality, is particularly easy to, in growth and breeding in 6~October, eat
The probability of thing poisoning and the mankind's is movable closely related.Body is caused to produce the food of poisoning, the typically food because of preservation
The thing resting period is long, and the too high high or food of environment temperature of storage is not thorough in heating process, causes bacterial spore to grow,
Cause bacterium amount reproduction in food, toxin is produced, so as to cause to be poisoned.Poisoner's symptom is stomachache, vomitted, diarrhoea.
Gemma caused by bacillus cereus is an important factor for causing food origin disease, gemma to high temperature, drying,
Ultraviolet, ionising radiation, toxic chemical substance and other unfavorable environmental factors have very strong resistivity.There is research
The amber reported from 25,000,000~40,000,000 years, which is neutralized in the salt solution inclusion for posting 2.5 hundred million years, successfully brings back to life gemma.Some waxes
The gemma of sample bacillus, more resistant to heat, can be subjected to cooking food than the gemma of mesophilic type bacillus subtilis and bacillus licheniformis
Many programs in product.Research reports that common food heating cooks (thermal shock) and can not kill the gemma of the bacterium, gemma meeting
Remaining is simultaneously germinateed, so as to cause the food of production contaminated.Therefore, the characteristic that bacillus cereus is difficult to kill is to cause its appearance
The main reason for easily causing food pollution and causing a disease.
Bacillus cereus has immune deficiency as a kind of important food-borne pathogenic microorganism, clinically main infection
People, such as old man, child, neonate.Also it can be infected by body fluid and cause bacteremia, endocarditis, meningitis and the eye of people
The diseases such as infection.Wherein, it is maximum with the social influence poisoned by food caused by bacillus cereus, and the waxy bud of social concerns
The reason for spore bacillus.The detection of bacillus cereus is divided into common detection methods and quick determination method.Common detection methods are,
Bacterium Zengjing Granule is carried out first, is isolated and purified, and passes through colony morphological observation, gram stain microscopy, physiological and biochemical test, medicine
The test for identification bacteriums such as quick experiment.Quick determination method is quickly examined with round pcr, immunological technique and enzyme reaction
Survey.Wang Zhenguo etc. establishes a kind of PCR method, is realized by expanding the hblA genes of bacillus cereus to waxy
The quick detection of bacillus, ELSEF etc. establish the detection method with real-time PCR detection bacillus cereus.Quick detection
The method of concrete operations is various, and detection speed is fast, high sensitivity, has reliability and accuracy, but sample preparation is more difficult, examines
Instrument level requirement height, the expensive reagents needed during survey.
2000, a kind of novel nucleic acid amplification method-ring mediated isothermal amplification (Loop- of Notomi et al. research and development
Mediated isothermal amplification, LAMP) technology, it is characterized in right for target gene specific designs 2
Specific primer, 6 regions of tested genome are demarcated, maintain under constant temperature 30 using strand displacement archaeal dna polymerase~
60min, you can complete nucleic acid amplification reaction.
The content of the invention
Goal of the invention：To overcome problem of the prior art, the invention provides one kind to utilize loop-mediated isothermal amplification technique
The primer and method of food-borne bacillus cereus are detected, can successful identification bacillus cereus.
Technical scheme：It is of the present invention to detect drawing for food-borne bacillus cereus using loop-mediated isothermal amplification technique
Thing, including outer primer and inner primer, the base sequence of outer primer are：
F3：5’-GATACATCTTCAATCGCTGG-3’
B3：5’-GGAAGTATACTAAATCACCTGG-3’；
The base sequence of inner primer is：
FIP：5’-TGAATCCACTGCAATCAAAACCATTTTTAAATGGTTCACCATACAGAACA-3’
BIP：5’-TGGTCATAAAGGCGCTCGTCTTTTCTAGTTTTTGTTTTAGAGCTCCAG-3’.
The main mode of infection of bacillus cereus is caused to infect and fallen ill by feed, and poisoner's symptom is abdomen
Bitterly, vomiting and diarrhoea.In the present invention, the food-borne species to bacillus cereus does not have restriction effect.
The bacterial strain of bacillus cereus different subtype, virulence gene carry very big otherness, present invention selection
Ent virulence genes as testing goal gene, by four sequence C P018935.1, CP012483.1, CP009628.1,
CP022445.1 is compared to obtain target conservative region, by the target conserved sequence in American National Biotechnology Information center
After (National Center of Biotechnology Information, NCBI) retrieval compares, it is found that this is listed 100
In individual comparison result, the genetic fragment of bacillus cereus is all belonged to, and does not occur the phenomenon met with other bacterial strains, therefore
The conserved sequence is applied to specific detection bacillus cereus.Further, devised for the conserved sequence, the present invention
LAMP primer, tested by covering primer more, it is found that above-mentioned primer has optimal Detection results.
Present invention also offers the method that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique, bag
Include：Extract the DNA of detected sample；
Using described DNA as template, LAMP reactions are carried out using described primer；
Reaction product is identified judge whether contain bacillus cereus in detected sample.
In LAMP reaction systems, the present invention for reaction specificity and all influential beet alkali concn of sensitivity, it is outer,
Inner primer concentration ratio, Mg2+Concentration, dNTPs concentration, reaction time and reaction temperature condition are optimized.LAMP is total to when reacting
4 specific primers are needed, the effect of outer primer is to react replacement process of the starting stage by chain, makes what inner primer synthesized
Chain departs from DNA profiling, produces dumbbell structure, since then during, the concentration of inner primer directly affects the expansion of reaction
Increasing Efficiency, the reaction process of incipient stage can be accelerated by properly increasing inner primer concentration, improve reaction efficiency, therefore, suitably interior,
Outer primer ratio, which can not only save primer dosage, can also make reaction reach best amplification efficiency.Mg in reaction system2+No
Only it is that to form the intermediate of stable base and the activation for enzymatic activity be necessary, the special of LAMP amplified reactions can be influenceed
The yield of property and amplified production, if Mg2+Concentration is too high, can largely increase the probability of primer mispairing, occurs non-specific
Amplification；If Mg2+Concentration is too low, cannot activate Bst DNA Polymerase activity.Glycine betaine is to make in reaction system
Existing for a kind of auxiliary element, glycine betaine is added without in experiment can also amplified reaction.But glycine betaine is denatured for DNA
Agent, be advantageous to the opening of DNA double spiral, can not only improve the specificity that primer and template strand combine but also DNA can be strengthened
Amplification efficiency, and have certain protection and stabilization to enzyme.DNTPs is also known as deoxyribonucleoside triphosphate (dNTP
Mixture), altogether comprising four parts:DATP, dGTP, dCTP and dTTP.It affects the specificity of reaction.If it
A kind of concentration of the concentration less than 5 μM or in remaining four part is less than the concentration of other parts, can cause LAMP amplifications mistake eventually
Only, the product amplified also just corresponding reduction.If dNTPs excessive concentration meeting and Mg2+Reaction is anti-so as to suppress LAMP
Should, product can also be reduced.
In summary, each reaction condition has been carried out preferably, LAMP reaction systems are：6-8mM Mg2+, 1.4-1.6mM
DNTPs, 1.0-1.2M glycine betaine, 0.2-0.3 μM of outer primer, 1.4-1.6 μM of inner primer, 1-1.5 μ L Bst archaeal dna polymerases,
2.5-3 μ L reaction buffers, 1-2 μ L gDNA, ddH2O is mended to 25 μ L.
It is further preferred that the LAMP reaction systems are：6mM Mg2+, 1.4mM dNTPs, 1.0M glycine betaines, 0.2 μM
Outer primer, 1.6 μM of inner primers, 1 μ L Bst archaeal dna polymerases, 2.5 μ L reaction buffers, 1 μ L gDNA, ddH2O is mended to 25 μ L.
The enzyme activity of Bst archaeal dna polymerases is 8-10U/ μ L.
Preferably, the temperature of LAMP reactions is 65-67 DEG C, more preferably 65 DEG C.
Preferably, the time of LAMP reactions is 60-65min, more preferably 60min.
After amplification terminates, by detect purpose fragment whether is amplified in reaction product come judge in detected sample whether
Contain bacillus cereus.Preferably, enter row agarose gel electrophoresis to reaction product to analyze and identify, the concentration of agarose is
2%.
Compared with prior art, beneficial effects of the present invention are：
The present invention is for the conserved sequence design primer of bacillus cereus ent virulence genes, a pair of LAMP reactions of going forward side by side
System optimizes, can stablize, high specific, highly sensitive detection bacillus cereus.
1st, specificity is higher：In above-mentioned four kinds of traditional diagnostic methods, meet high specific, it is quick the characteristics of be tradition
Molecular biology method.Two of traditional molecular biology method, the only inhereditary material (DNA) of the detected microorganism of locking
Detected in region；This method is that six regions that the inhereditary material (DNA) for being detected microorganism is locked with 4 primers are carried out
Detection, specificity are higher.
2nd, sensitivity is more preferable：Identification, Tissue pathological diagnosis method, immunology detection side are separately cultured in the tradition of bacterium
In method, conventional molecular biological detection method, the sensitivity of the detection method of molecular biology is highest, and this research method
Sensitivity in contrast test PCR, within the shorter reaction time, sensitivity is at least higher by a quantity
Level.
3rd, experimentation cost is lower：In this method detection process, carry out, therefore need not hold high under the conditions of tangible water bath with thermostatic control
Expensive variable temperature unit, in diagnostic result, it can carry out sentencing for result by naked eyes or the simple indicator of addition
Disconnected, the cost of experiment is all lower than other traditional approach.
4th, quick detection time：In above-mentioned four kinds of detection methods, the detection speed of traditional molecular biology method and
Cycle is most fast, and this method is also one kind in molecular biology method, therefore also maintains diagnosis of molecular biology fast cycle
The characteristics of short.When observing result, the time of cost than traditional molecular biology method faster, in contrast and polymerase chain
In the contrast test of reaction, PCR is about completed in 1.5h hours, and the reaction time of this method only needs 60 points
Clock, so detection cycle and speed are faster.
Brief description of the drawings
Fig. 1 is glycine betaine (betaine) concentration optimization (M), M：DL2000 DNA Marker, N：Negative control；
Fig. 2 is Mg2+Concentration optimization (mM), M：DL2000 DNA Marker, * are the condition of the optimization of selection, N：It is negative right
According to；
Fig. 3 is dNTPs concentration optimizations (mM), M：DL2000 DNA Marker, * are the condition of the optimization of selection, N：It is negative
Control；
Fig. 4 is interior outer primer ratio optimization (inner primer μM), M：DL2000 DNA Marker, * are the bar of the optimization of selection
Part, N：Negative control；
Fig. 5 is that reaction temperature optimizes (DEG C), M：DL2000 DNA Marker, * are the condition of the optimization of selection, N：It is negative
Control；
Fig. 6 is to optimize (min) in the reaction time, M：DL2000 DNA Marker, * are the condition of the optimization of selection, N：It is negative
Control；
Fig. 7-1 be LAMP specific detections a part of result, M：DL2000 DNA Marker, swimming lane 1-10 divide successively
It is not：Bacillus cereus CGMCC 1.195, bacillus cereus CMCC (B) 63303, Yersinia ruckeri, vibrio parahaemolytious,
Candida albicans bacterium, Salmonella choleraesuls, Friedlander's bacillus, staphylococcus aureus, micrococcus lysodeikticus, withered grass gemma
Bacillus；
Fig. 7-2 be LAMP specific detections another part result, M：DL2000 DNA Marker, swimming lane 1,2,11-18
It respectively is：(antibiotic reflects for bacillus cereus CGMCC 1.195, bacillus cereus CMCC (B) 63303, Escherichia coli
Singling), micrococcus luteus, Edwardsiella tarda, salmonella CMCC 50774, salmonella CMCC 50001, Salmonella
Bacterium CMCC 50004, salmonella CGMCC 50071, salmonella CGMCC 500115, N：Negative control；
Fig. 8-1 is that the sensitivity of LAMP method detects (ng/ μ L), M：DL2000DNA Marker, N：Negative control, path
1-7 DNA concentration respectively is 7.75,7.75 × 10-1、7.75×10-2、7.75×10-3、7.75×10-4、7.75×10-5、7.75×10-6, * expression minimum detection limits；
Fig. 8-2 is that the sensitivity of PCR method detects (ng/ μ L), M：DL2000 DNA Marker, N：Negative control, path
1-7 DNA concentration respectively is 7.75,7.75 × 10-1、7.75×10-2、7.75×10-3、7.75×10-4、7.75×10-5、7.75×10-6, * expression minimum detection limits；
Fig. 9 is specific outcome figure in comparative example 1；
Figure 10 is specific outcome figure in comparative example 2；
Figure 11 is the techniqueflow chart of the inventive method.
Embodiment
With reference to specific embodiment, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention, after the present invention has been read, various equivalences of the those skilled in the art to the present invention
The modification of form falls within the application appended claims limited range.
In the specific embodiment of the invention, material therefor is as follows：
1 main agents：
Triphosphate deoxy-nucleotide (dNTPs, 25mM), glycine betaine (betaine), the type digestive ferments of DNase I are purchased from raw work
Bioengineering (Shanghai) limited company；Magnesium ion (Mg2+, 100mM), reaction buffer (10 × thermpol reaction
Buffer Mg containing 2mM2+) with Bst DNA Polymerase be purchased from knob Great Britain biotechnology (Beijing) Co., Ltd；High-purity PCR
Template reagent preparation box is purchased from precious bioengineering (Dalian) Co., Ltd；2 pairs of LAMP primers synthesize in raw work bioengineering (on
Sea) limited company；LB fluid nutrient mediums are purchased from Qingdao Hai Bo Bioisystech Co., Ltd；2 × PCR Master Mix, fine jade
Lipolysaccharide, 50 × TAE buffer solutions, DL 2000DNA Maker, the type nucleic acid dyes of Gold view I are purchased from Tiangeng biochemical technology (north
Capital) Co., Ltd；Other reagents are that autogamy or analysis are pure.
2 main bacteria seeds
Table 1 tests bacterial strain uses therefor
Note：Above bacterial strain derives from Amphixenosis research institute of Jilin University, bacterium group laboratory, people in the art
Member can also be bought according to strain number from company or preservation mechanism obtains.
3 instrument and equipments
Electronic analytical balance (BSA4202S-CW, Beijing Sai Duolisi companies), miniature high-speed refrigerated centrifuge (Sorvall
Legend Micro 21R, match Mo Feishier company), 2720 type PCR amplification instruments (ABI companies), DYY-8C type electrophoresis apparatuses (Beijing
61 instrument plants), microplate spectrophotometer (Epoch, BioTek company), chemiluminescence gel imaging system (1708195,
Bole company), thermostat water bath (XO-8D, Nanjing Xian Ou companies), 4 DEG C of refrigerators (BCD-271TMBA, company of Haier), -20 DEG C
Refrigerator-freezer (BD/BC-415DKMQ415, beautiful company), superclean bench (BCM-1300, Suzhou purification Co., Ltd).
The preparation of 4 common agents
The preparation of 5M glycine betaines (betaine) solution：Glycine betaine powder 5.8g is weighed, adds 8mL sterilizings ddH20 shakes up, and fills
Divide dissolving, last constant volume to 10mL, the filtering of 0.22mm filters, dispense into sterile 1.5mL centrifuge tubes, -20 DEG C save backup.
The preparation of LB fluid nutrient mediums：LB powder 2.5g are weighed, add 80mL sterilizings ddH2O is dissolved by heating, last constant volume
To 100mL, PH to 7.4 is adjusted, is dispensed to test tube, 121 DEG C of autoclaving 20min.
The preparation of tryptose soya agar culture medium：Tryptone 15.0g, soy peptone 5.0g, chlorine are weighed respectively
Change sodium 5.0g, nutrient agar 15.0g, be added sequentially to 1000mL sterilizings ddH2Heated in O, fully dissolving, adjust PH to 7.4,
121 DEG C of autoclaving 20min, when waiting temperature is down to 60 DEG C, culture medium is poured into sterilizing flat board in sterile super-clean bench, it is cold
It is standby.
Embodiment 1
The rejuvenation and culture of 1 bacterium
Before culture, each strain deposited of going bail for carries out line rejuvenation on tryptose soya agar flat board, is put into constant temperature training
Support 37 DEG C of culture 12h of case.It is seeded to oese picking single bacterium colony in LB fluid nutrient mediums, 37 DEG C of shaken cultivation 18-24h.
It is sub-packed in the 2.0mL centrifuge tubes for having added qs glycerin, -20 DEG C of preservations.
The extraction of 2 bacterial genomes
Take and dispense standby 2.0mL centrifuge tubes, 12000rpm/min, centrifuge 1min, discard supernatant, with sterilizing ddH2O
Washing thalline twice, then dissolves thalline with 50 μ L sterilized waters.Explanation extraction according to high-purity pcr template reagent preparation box is corresponding
The genome of bacterium, -20 DEG C save backup.
The design and processing of 3 bacillus cereus LAMP primers
In NCBI net search in Website bacillus cereus ent virulence gene (accession number：CP018935.1、CP012483、
CP009628.1, CP022445.1), above-mentioned ent gene orders are contrasted, find out conserved sequence, then for conserved sequence
Design a series of LAMP primers, including a pair of inner primers and a pair of outer primers.Primer sequence is shown in Table 2.
The LAMP primer sequence of table 2
Note α：Marked according to bacillus cereus ent genes (GenBank accession number CP022445.1 please check accession number)
Primer location.
LAMP primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the explanation of report is synthesized according to primer
Book, by 12000 × g of centrifuge tube equipped with primer, 30s is centrifuged, the primer for being attached on tube wall is focused on ttom of pipe.Add ormal weight
Sterilizing ddH2O, final concentration of 100 μM of inner primer dilution, final concentration of 10 μM of outer primer dilution.
4 LAMP detect the condition optimizing of bacillus cereus
LAMP reaction systems：1 μ L Bst DNA Polymerase (8U/ μ L), glycine betaine (betaine), 2.5 μ L10 ×
Thermpol reaction buffer, Mg2+, dNTPs, 1 μ L genomic templates (gDNA) inner primer (BIP and FIP) and draw outside
Thing (B3 and F3), finally with sterilizing ddH2O complements to 25 μ L, and reaction condition is 65 DEG C of water-bath 60min.Wherein for the spy of reaction
The opposite sex and all influential beet alkali concn of sensitivity, outer inner primer concentration ratio, Mg2+Concentration, dNTPs concentration, the reaction time and
Reaction temperature condition optimizes.
The optimization of 4.1 glycine betaines (betaine)
Glycine betaine concentration optimization condition and range：0~1.4M, per mono- gradient of 0.2M.In reaction system, except beet alkali concn
Outer other components concentration is constant, including 8mM Mg2+, 1.6mM dNTPs, 0.2 μM of outer primer (B3 and F3), 1.6 μM of inner primers
(BIP and FIP), after 65 DEG C of water-bath 60min, LAMP reactions are terminated in 80 DEG C of water-bath 5min inactivation Bst DNA Polymerase,
With 2% Ago-Gel, add after nucleic acid dye fully mixes with 5 μ L response samples, progress nucleic acid electrophoresis.
4.2Mg2+The optimization of concentration
Mg2+Concentration optimization condition and range：1.0~8.0mM, per mono- gradient of 1.0mM.In reaction system, except Mg2+Concentration
Outer other components concentration is constant, including 1.6mM dNTPs, 1.0M glycine betaines (betaine), 0.2 μM of outer primer (B3 and F3),
1.6 μM of inner primers (BIP and FIP) are whole in 80 DEG C of water-bath 5min inactivation Bst DNA Polymerase after 65 DEG C of water-bath 60min
Only LAMP reacts, and with 2% Ago-Gel, adds after nucleic acid dye fully mixes with 5 μ L response samples, progress nov nucleic acid
Swimming.
The optimization of 4.3 dNTPs concentration
DNTPs concentration optimization scopes：1.0~2.4mM, per mono- gradient of 0.2mM.In reaction system, in addition to dNTPs concentration
Other components concentration is constant, including 8mM Mg2+, 1.0M glycine betaines, 0.2 μM of outer primer (B3 and F3), 1.6 μM of inner primers (BIP and
FIP), after 65 DEG C of water-bath 60min, LAMP reactions are terminated in 80 DEG C of water-bath 5min inactivation Bst DNA Polymerase, with 2%
After Ago-Gel, addition nucleic acid dye and 5 μ L response samples fully mix, nucleic acid electrophoresis is carried out.
4.4 inside and outside primer concentrations than optimization
Inner primer concentration optimization scope：0.2~1.6 μM, every 0.2 μM of gradient.Inside and outside primer concentration is than optimization model
Enclose：1:1、2:1、3:1、4:1、5:1、6:1、7:1 and 8:1.In reaction system, other groups in addition to inner primer (BIP and FIP) concentration
Divide concentration constant, including 8mM Mg2+, 1.0M glycine betaines, 1.4mM dNTPs, 0.2 μM of outer primer (B3 and F3), 65 DEG C of water-baths
After 60min, LAMP reactions are terminated in 80 DEG C of water-bath 5min inactivation Bst DNA Polymerase, with 2% Ago-Gel, are added
Enter nucleic acid dye and after 5 μ L response samples fully mix, carry out nucleic acid electrophoresis.
4.5 the optimization of reaction temperature
Reaction temperature optimization range：55~80 DEG C, every 5 DEG C of gradients.25 μ L reaction systems, including 2.5 μ L10 ×
Thermpol reaction buffer, 1 μ LBst DNA Polymerase, 8mM Mg2+, 1.0M glycine betaines, 1.4mM
DNTPs, 0.2 μM of outer primer (B3 and F3), 1.6 μM of inner primers (BIP and FIP), the reaction time is 60min.Finally in 80 DEG C of water
Bathe 5min inactivation Bst DNA Polymerase and terminate LAMP reactions, with 2% Ago-Gel, add nucleic acid dye and 5 μ L
After response sample fully mixes, nucleic acid electrophoresis is carried out.
The optimization in 4.6 reaction time
Reaction time optimization range：30~80min, per mono- gradient of 10min.25 μ L reaction systems, including 2.5 μ L10 ×
Thermpol reaction buffer, 1 μ LBst DNA Polymerase, 8mM Mg2+, 1.0M glycine betaines, 1.4mM
DNTPs, 0.2 μM of outer primer (B3 and F3), 1.6 μM of inner primers (BIP and FIP), reaction temperature is 65 DEG C.Finally in 80 DEG C of water-baths
5min inactivation Bst DNA Polymerase terminate LAMP reactions, with 2% Ago-Gel, add nucleic acid dye and 5 μ L are anti-
After answering sample fully to mix, nucleic acid electrophoresis is carried out.
Testing result is shown in Fig. 1-Fig. 6, and the optimum condition of optimization is：Glycine betaine 1.0M, Mg2+6mM, dNTPs1.4mM, inside draw
1.6 μM of thing, 0.2 μM of outer primer, reaction temperature are 65 DEG C, reaction time 60min.
Embodiment 2LAMP detects the specific detection of bacillus cereus
To 18 plants of bacteriums listed by table 1 using RNA isolation kit extraction genomic DNA, with the ddH that sterilizes2O is that template sets feminine gender
Control, according to 25 μ L reaction systems after optimization carry out LAMP amplifications (2.5 μ L10 × thermpol reaction buffer, 1
μ LBst DNA Polymerase, 6mM Mg2+, 1.0M glycine betaines, 1.4mM dNTPs, 0.2 μM of outer primer (B3 and F3), 1.6 μM
Inner primer (BIP and FIP), 65 DEG C of water-bath 60min), the specificity for examining LAMP to react.After reaction terminates, in 80 DEG C of water-baths
5min inactivates Bst DNA Polymerase, and detect by an unaided eye klebsiella pneumoniae pipe and negative findings control tube ratio first
Compared with observation tube bottom after simple centrifugation, carrying out preliminary judged result.With 2% Ago-Gel, nucleic acid dye and 5 μ L are added
After response sample fully mixes, nucleic acid electrophoresis is carried out.
As a result can specific detection bacillus cereus referring to Fig. 7-1 and 7-2, invention of the present invention.
The comparison of the bacillus cereus sensitivity of embodiment 3 detection
1 LAMP detects bacillus cereus sensitivity
The bacillus cereus genomic DNA extracted in a small amount of 2 step is taken, with the Take3 journeys of microplate spectrophotometer
The initial concentration of sequence and Instrument measuring (Gene companies) genome.Then with sterilizing ddH2O carries out 10 times of dilutions to genome, altogether
Dilute 7 gradients.
With reaction system (2.5 μ L10 × thermpol reaction buffer, 1 μ LBst DNA optimized
Polymerase, 6mM Mg2+, 1.0M glycine betaines, 1.4mM dNTPs, 0.2 μM of outer primer (B3 and F3), 1.6 μM of inner primer (BIP
And FIP), 65 DEG C of water-bath 60min) to the genome progress specific amplification of 7 gradients.After reaction terminates, in 80 DEG C of water-baths
5min inactivation Bst DNA Polymerase.With 2% Ago-Gel, add 1 μ 6 × loading of L buffer's and 5 μ L
After sample fully mixes, nucleic acid electrophoresis is carried out, observation, analysis result under gel imager.
2 conventional PCR methods detect bacillus cereus sensitivity
Consulting literatures (Davide Porcellato, Judith Narvhus, Siv Borghild
Skeie.Detection and quantification of Bacillus ceres group in milk by droplet
Digital PCR [J] .Journal of Microbiological Methods, 2016.), according to bacillus cereus cer
Gene designs one couple of PCR primers, primer sequence：
Upstream (SEQ ID NO.5) 5 '-GCTAAAAGGTGTACTTAGCTTAGG-3 '；
Downstream (SEQ ID NO.6) 5 '-TATATACATTATGCGTCATCAC-3 '.PCR reaction systems are 25 μ L, including 2
The μ L of × PCR Master Mix 12.5,1.0 μ L sense primers, 1.0 μ L anti-sense primers, dNTPs1.4mM, 1 μ L gDNA, finally
With sterilizing ddH2O is mended to 25 μ L.Reaction condition is as follows：94℃5min；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 30s, 35 circulations；72
℃7min.Reaction carries out 1.0% gel electrophoresis after terminating.Purpose fragment size is expanded in 297bp.
As a result 7.75 × 10 are limited to referring to Fig. 8-1, Fig. 8-2, the lowest detection of LAMP method-4Ng/ μ L, conventional PCR method
Lowest detection be limited to 7.75 × 10-2ng/μL。
To sum up, this experiment be based on ent virulence genes, and bacillus cereus LAMP experiments are required draws for successful design
Thing.LAMP reaction systems are optimized, the determination of final reaction system is：6mM Mg2+, 1.4mM dNTPs, 1.0M glycine betaines, 0.2
μM outer primer (B3 and F3), 1.6 μM of inner primers (BIP and FIP), 1 μ L Bst DNA Polymerase (8U/ μ L), 2.5 μ L10
× thermpol reaction buffer, 1 μ L gDNA, with sterilizing ddH2O is mended to 25 μ L, and reaction condition is 65 DEG C of water-baths
60min.As a result show：LAMP method detection bacillus cereus genome minimum detection limit is 0.775pg/ μ L, is normal PCR
100 times of method, detection time shortens nearly 1h, has very high sensitivity and stability.
Comparative example 1
In NCBI net search in Website bacillus cereus ces virulence gene (accession number：JN112796.1、CP001166.1、
AP007210.1, AY691650.1), ces gene orders are contrasted, find out conserved sequence, then design for conserved sequence
A series of LAMP primers, including a pair of inner primers and a pair of outer primers.Primer sequence is shown in Table 3.
The LAMP primer sequence of table 3
Note b：The primer location marked according to bacillus cereus ces genes (GenBank accession number JN112796.1).
Using this primer, during carrying out specificity experiments, there is detected bacillus cereus without the positive
As a result, illustrate that primer specificity is undesirable.As a result in Fig. 9.
(note:1st, 2 be bacillus cereus, and 3-5 is other non-bacillus cereus strains, N：Negative control)
Comparative example 2
In NCBI net search in Website bacillus cereus ces virulence gene (accession number：JN112795.1、DQ889676.1、
AP007210.1, CP001179.1), ces gene orders are contrasted, find out conserved sequence, then design for conserved sequence
A series of LAMP primers, including a pair of inner primers and a pair of outer primers.Primer sequence is shown in Table 4.
The LAMP primer sequence of table 4
Note α：The primer location marked according to bacillus cereus ces genes (GenBank accession number JN112795.1).
Using this primer, during carrying out specificity experiments, although detected bacillus cereus has positive findings, but
It is during specific detection, other strains also have positive findings, illustrate that primer specificity is undesirable.As a result in Figure 10 (notes:
Swimming lane 1,2 is bacillus cereus, and 3-5 is other non-bacillus cereus strains, N：Negative control).
Sequence table
<110>Jiangsu Polytechnic College of Agriculture and Forestry
<120>The method and primer of food-borne bacillus cereus are detected using loop-mediated isothermal amplification technique
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gatacatctt caatcgctgg 20
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ggaagtatac taaatcacct gg 22
<210> 3
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tgaatccact gcaatcaaaa ccatttttaa atggttcacc atacagaaca 50
<210> 4
<211> 48
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tggtcataaa ggcgctcgtc ttttctagtt tttgttttag agctccag 48
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gctaaaaggt gtacttagct tagg 24
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
tatatacatt atgcgtcatc ac 22
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ttccagtact gcctgacg 18
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
cactgcaaca catttggc 18
<210> 9
<211> 54
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ggataaaggg aagcaagagg agttattttt acataatata tgccgtatct tctg 54
<210> 10
<211> 44
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ccttcgatgt ccacaaacgg aattttatag cggtgcaaaa gtcc 44
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
atagcggtgc aaaagtcc 18
<210> 12
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
accataaccc cttttggtt 19
<210> 13
<211> 47
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
cgatgtccac aaacggaact ttttttccta gggaaaaatg aaacaac 47
<210> 14
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
aagggaagca gaggagttag tttttttcca gtactgcctg acg 43
Claims (6)
1. a kind of primer that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique, including outer primer and interior draw
Thing, it is characterised in that the base sequence of outer primer is：
F3：5’-GATACATCTTCAATCGCTGG-3’
B3：5’-GGAAGTATACTAAATCACCTGG-3’；
The base sequence of inner primer is：
FIP：5’-TGAATCCACTGCAATCAAAACCATTTTTAAATGGTTCACCATACAGAACA-3’
BIP：5’-TGGTCATAAAGGCGCTCGTCTTTTCTAGTTTTTGTTTTAGAGCTCCAG-3’.
A kind of 2. method that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique, it is characterised in that including：
Extract the DNA of detected sample；
Using described DNA as template, LAMP reactions are carried out using the primer described in claim 1；
Reaction product is identified judge whether contain bacillus cereus in detected sample.
3. the method according to claim 2 that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique,
Characterized in that, LAMP reaction systems are：6-8mM Mg2+, 1.4-1.6mM dNTPs, 1.0-1.2M glycine betaines, 0.2-0.3 μM
Outer primer, 1.4-1.6 μM of inner primer, 1-1.5 μ L Bst archaeal dna polymerases, 2.5-3 μ L reaction buffers, 1-2 μ L gDNA,
ddH2O is mended to 25 μ L.
4. the method according to claim 2 that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique,
Characterized in that, the temperature of LAMP reactions is 65-67 DEG C.
5. the method according to claim 2 that food-borne bacillus cereus is detected using loop-mediated isothermal amplification technique,
Characterized in that, the time of LAMP reactions is 60-65min.
6. the method according to claim 2 that food-borne Klebsiella Pneumoniae is detected using loop-mediated isothermal amplification technique,
Analyzed and identified characterized in that, entering row agarose gel electrophoresis to reaction product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710804909.6A CN107475401B (en) | 2017-09-08 | 2017-09-08 | Method and primer for detecting food-borne bacillus cereus by using loop-mediated isothermal amplification technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710804909.6A CN107475401B (en) | 2017-09-08 | 2017-09-08 | Method and primer for detecting food-borne bacillus cereus by using loop-mediated isothermal amplification technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107475401A true CN107475401A (en) | 2017-12-15 |
| CN107475401B CN107475401B (en) | 2021-03-19 |
Family
ID=60584637
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710804909.6A Active CN107475401B (en) | 2017-09-08 | 2017-09-08 | Method and primer for detecting food-borne bacillus cereus by using loop-mediated isothermal amplification technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107475401B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996266A (en) * | 2020-06-05 | 2020-11-27 | 江苏海洋大学 | Specific forward and reverse primers, probe and detection kit for vomit-causing bacillus cereus and application of specific forward and reverse primers and probe |
| CN112575097A (en) * | 2020-10-12 | 2021-03-30 | 南开大学 | Liquid phase chip for detecting bacillus cereus and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200619385A (en) * | 2004-12-03 | 2006-06-16 | Of Food And Drug Analysis Dept Of Health Bureau | Multiplex primers for rapid and highly specific detection of diarrheal enterotoxin genes in bacillus cereus |
| CN101402997A (en) * | 2008-11-06 | 2009-04-08 | 中华人民共和国天津出入境检验检疫局 | Reagent kit and method for detecting bacillus cereus with ring mediated isothermality amplification method |
| CN101831493A (en) * | 2009-11-06 | 2010-09-15 | 武汉工业学院 | Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method |
| CN103725751A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Method for highly-specific highly-sensitive detection of vomitoxin-secreting bacillus cereus |
| WO2014148877A1 (en) * | 2013-03-22 | 2014-09-25 | 나노바이오시스 주식회사 | Primer set for detecting food poisoning, pcr apparatus using same, and method for detecting food poisoning using same |
| CN106434902A (en) * | 2016-08-31 | 2017-02-22 | 北京卓诚惠生生物科技股份有限公司 | Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof |
| CN106434889A (en) * | 2015-09-02 | 2017-02-22 | 上海旺旺食品集团有限公司 | Method for rapidly detecting bacillus cereus at constant temperature, primers used for method and applications of primers |
-
2017
- 2017-09-08 CN CN201710804909.6A patent/CN107475401B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200619385A (en) * | 2004-12-03 | 2006-06-16 | Of Food And Drug Analysis Dept Of Health Bureau | Multiplex primers for rapid and highly specific detection of diarrheal enterotoxin genes in bacillus cereus |
| CN101402997A (en) * | 2008-11-06 | 2009-04-08 | 中华人民共和国天津出入境检验检疫局 | Reagent kit and method for detecting bacillus cereus with ring mediated isothermality amplification method |
| CN101831493A (en) * | 2009-11-06 | 2010-09-15 | 武汉工业学院 | Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method |
| CN103725751A (en) * | 2012-10-10 | 2014-04-16 | 苏州四同医药科技有限公司 | Method for highly-specific highly-sensitive detection of vomitoxin-secreting bacillus cereus |
| WO2014148877A1 (en) * | 2013-03-22 | 2014-09-25 | 나노바이오시스 주식회사 | Primer set for detecting food poisoning, pcr apparatus using same, and method for detecting food poisoning using same |
| CN106434889A (en) * | 2015-09-02 | 2017-02-22 | 上海旺旺食品集团有限公司 | Method for rapidly detecting bacillus cereus at constant temperature, primers used for method and applications of primers |
| CN106434902A (en) * | 2016-08-31 | 2017-02-22 | 北京卓诚惠生生物科技股份有限公司 | Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof |
Non-Patent Citations (6)
| Title |
|---|
| NGAMWONGSATIT P ET AL: "Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers", 《INT J FOOD MICROBIOL》 * |
| 周巍 等: "环介导等温扩增技术检测酸乳中蜡样芽孢杆菌", 《乳业科学与技术》 * |
| 谭贵良 等: "《现代分子生物学及组学技术在食品安全检测中的应用》", 30 June 2014, 中山大学出版社 * |
| 贾雅菁 等: "实时荧光环介导等温扩增技术检测牛乳中的蜡样芽孢杆菌", 《食品科学》 * |
| 邵秀玲 等: "《植物病原生物现代检测技术及应用》", 31 October 2015, 中国质检/标准出版社 * |
| 齐哲: "环介导等温扩增技术快速检测蜡样芽孢杆菌研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996266A (en) * | 2020-06-05 | 2020-11-27 | 江苏海洋大学 | Specific forward and reverse primers, probe and detection kit for vomit-causing bacillus cereus and application of specific forward and reverse primers and probe |
| CN112575097A (en) * | 2020-10-12 | 2021-03-30 | 南开大学 | Liquid phase chip for detecting bacillus cereus and application |
| CN112575097B (en) * | 2020-10-12 | 2022-05-10 | 南开大学 | Liquid phase chip for detecting bacillus cereus and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107475401B (en) | 2021-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Strayer-Scherer et al. | Recombinase polymerase amplification assay for field detection of tomato bacterial spot pathogens | |
| CN102154473B (en) | Gene chip and applications thereof in detection of aquatic pathogenic microorganism | |
| CN104450940B (en) | A kind of nucleotide sequence and detection method and detection kit for detecting Listeria Monocytogenes | |
| CN101418335A (en) | Reagent for detection of pathogenic hydrophila gingivalis and detection method thereof | |
| CN107365869A (en) | The method and primer of food-borne Klebsiella Pneumoniae are detected using loop-mediated isothermal amplification technique | |
| CN109554449B (en) | Multiplex PCR method capable of simultaneously detecting 7 virulence genes of aeromonas | |
| Sullia et al. | General microbiology | |
| CN101967516B (en) | Vibrio cholerae typing and virulence gene detection kit and detection method | |
| CN103898227A (en) | Nucleotide specific to cronobacter O antigen and application of nucleotide | |
| CN102676664A (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| CN102154497A (en) | M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella | |
| CN107475401A (en) | The method and primer of food-borne bacillus cereus are detected using loop-mediated isothermal amplification technique | |
| CN107022638A (en) | The LAMP primer group of quick detection salmonella and its application | |
| CN105385773B (en) | A kind of method detecting Pseudomonas fluorescens and its kit and primer | |
| CN102424862B (en) | Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila | |
| CN1312475C (en) | Molecular detection method for fusarium toxin | |
| CN101586157A (en) | Diagnostic kit for Streptococcus iniae molecule and detection method | |
| CN107988330A (en) | The dual-PCR method of Listeria monocytogenes and listeria ivanovii is detected at the same time | |
| CN109811073A (en) | Double PCR early stage quickly detects primer and its application of Streptococcusagalactiae and Streptococcus iniae | |
| KR101752274B1 (en) | Primer set for high sensitive real-time multiplex loop-mediated isothermal amplification reaction for determining type of shiga toxin genes stx1 and stx2 of Enterohemorrhagic Escherichia coli, and method for determining type of shiga toxin genes of Enterohemorrhagic Escherichia coli using the same | |
| CN108342499B (en) | A pair of while quickly detection Streptococcusagalactiae and Streptococcus iniae primer and its application | |
| CN105256028B (en) | The nucleotide special to citric acid bacillus 017 and O39 and its application | |
| CN103981266A (en) | Method for detecting coliforms in dairy products and primer used thereby | |
| Tabashsum et al. | Current Practices for Isolation, Identification and Characterization of Campylobacter in Foods and Recommendations for Future | |
| JP5134071B2 (en) | Method for avoiding inhibition in nucleic acid amplification reaction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |